Dental plaque is a complex oral biofilm comprised of a diverse microbial community, which necessitates that bacteria coordinate their behavior and function based on the environment and other organisms in the biofilm. In the oral pathogen Aggregatibacter actinomycetemcomitans this intimate cell-to- cell communication potentially occurs via the quorum sensing circuit dependent upon the soluble signaling molecule autoinducer-2 (AI-2). Previous results suggest that the response circuits of A. actinomycetemcomitans differ from the established paradigms for AI-2 signaling. Thus, we hypothesize that the AI-2-mediated quorum sensing response circuit in A. actinomycetemcomitans is tailored towards the specific survival strategies employed by this organism in the oral biofilm and contributes to its in vivo persistence and virulence through AI-2 regulation of iron acquisition. To address this hypothesis, we will further characterize the molecular mechanisms of AI-2-dependent quorum sensing in A. actinomycetemcomitans. Our first specific aim is to determine the importance of AI-2-dependent quorum sensing in the persistance and virulence of A. actinomycetemcomitans. Since periodontal diseases are mixed bacterial infections of the oral cavity, it is theorized that populational shifts in the biofilm that leads to overrepresentation of certain organisms, rather than their absence or presence, is a major contributing factor to the onset and progression of oral diseases. Using the Baker model of periodontitis, we will determine if AI-2-mediated quorum sensing is important in vivo during single infections with either wild type or AI-2 receptor-deficient strains of A. actinomycetemcomitans. Our second specific aim is to identify the component that links AI-2 detection to downstream gene regulation. Initial studies suggest that the two- component system QseBC may link detection and gene regulation. To address our hypothesis, we will determine if QseBC regulates biofilm formation in A. actinomycetemcomitans. This will be accomplished by constructing QseBC mutants and analyzing their ability to form biofilms in vitro. We will also determine if QseBC influences the expression of AI-2-regulated genes such as iron acquisition by utilizing quantitative PCR. Public Health Relevance: Determining the extra- and intracellular physiologic roles of AI-2 will provide rationale for the development of novel quorum sensing inhibitors as a way to prevent the growth of A. actinomycetemcomitans biofilms associated with oral diseases as well as other diseases (e.g. cardiovascular disease). Furthermore, innovative methods to control biofilm formation in the oral cavity would have a major impact on healthcare costs